CN106834472A - BCR diversity detection kit and application - Google Patents
BCR diversity detection kit and application Download PDFInfo
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Abstract
The present invention provides a kind of detection multifarious kits of BCR, and the kit includes one group of multiple PCR primer, and the multiple PCR primer includes sense primer and anti-sense primer, sense primer by with SEQ ID NO:1~SEQ ID NO:The one-to-one one group of sequence composition of nucleotide sequence shown in 13, the sequence in sense primer is than SEQ ID NO:1~SEQ ID NO:Corresponding sequence in 13 is more or few 0~3 nucleotides, anti-sense primer by with SEQ ID NO:14~SEQ ID NO:The one-to-one one group of sequence composition of nucleotide sequence shown in 17, the sequence in anti-sense primer is than SEQ ID NO:14~SEQ ID NO:Corresponding sequence in 17 is more or lacks 0~3 nucleotides.Using the kit, the BCR sequences of people can be efficiently obtained, obtain the BCR CDR3 sequences of human specific, the recall rate of low copy number B cell clone can be improve.
Description
Related application
It is on 08 14th, 2015 the applying date that the application is, entitled " it is micro- that one kind builds leukaemia based on high-flux sequence
The division of the Chinese patent application 201510500391.8 of the multiple PCR primer and method in small residual BCR libraries ".
Technical field
The invention belongs to biology field, it is related to gene detecting kit, especially, is related to a kind of BCR diversity
Detection kit and application.
Background technology
The antigentic specificity of T/B lymphocytes is largely by T/B lymphocyte receptor CDR3 Region amino acids
Sequence is determined.Upper a large amount of V, J genetic fragments of T/B cytogenes seat can produce various various rearrangements in the synthesis of acceptor, in V-
Nucleotides between J, V-D and D-J conjugant is independent of template insertion or deletes, this further increase similar with high frequency closedown
Acceptor potential diversity.This potential diversity of acceptor is difficult to randomly generate identical TCRB/IGH chain CDR3 sequences,
So that each CDR3 sequence effectively becomes a unique tags for T/B cell clones.
Therefore constituted for the sequence in bone-marrow-derived lymphocyte IGH gene C DR3 regions and detected, can be very good to reflect BCR
The composition and response situation in immune group storehouse.
At present, the acquisition of the BCR genes of people or analysis method still have much room for improvement.
The content of the invention
The invention provides a kind of kit comprising the multiple PCR primer that can be used in expanding people BCR and application.
In a first aspect, the invention provides one kind detection multifarious kits of BCR, the kit is multiple comprising one group
PCR primer, the multiple PCR primer include sense primer and anti-sense primer, the sense primer by with SEQ ID NO:1~SEQ
ID NO:The one-to-one one group of sequence composition of nucleotide sequence shown in 13, the sequence in the sense primer is than SEQ ID
NO:1~SEQ ID NO:Corresponding sequence in 13 is more or lacks 0~3 nucleotides；The anti-sense primer by with SEQ ID NO:14
~SEQ ID NO:The one-to-one one group of sequence composition of nucleotide sequence shown in 17, the sequence ratio in the anti-sense primer
SEQ ID NO:14~SEQ ID NO:Corresponding sequence in 17 is more or lacks 0~3 nucleotides.Herein, unless otherwise noted,
" base " is equal to " nucleotides ", such as, during counting, 1 nucleotides is represented with 1bp." more or few 0~3 nucleotides " be
Hold many or few 0~3 nucleotides in the 3 ' of correspondence primer.Inventor in design and experiment sieving multiple PCR primer group, in surprise
Ground finds, extreme position ground, interval or does not extend continuously and/or truncates SEQ ID NO:1~SEQ ID NO:13 and/or SEQ
ID NO:14~SEQ ID NO:At least one sequence in 17 is no more than 3nt, and the multiple PCR primer group for being formed still may be used
Effectively to obtain target sequence.
PCR multi-primerses in the kit are variable region V area (variable area) and J area of the inventor for people BCR
What many secondary designs of (joining areas, bonding pad) sequence characteristic, test of many times determined, this group of sequence can same reaction system,
Target sequence is expanded without interfering with each other.Enough BCR gene sequences can be efficiently obtained comprising this group of kit of multi-primerses
Row, can be used in the multifarious detection and analysis of BCR.
The kit of this aspect of the invention, can also have at least one of following additional technical feature：
Extension or truncation can be continuous nucleotide, or discontinuous kernel thuja acid, can in terminal extension or
Truncating can also be in centre extension or truncation.Embodiments in accordance with the present invention, " more or few 0~3 nucleotides " is to draw in correspondence
Hold many or few 0~3 nucleotides in the 3 ' of thing.
Embodiments in accordance with the present invention, terminal extension or truncation in primer sequence.
Embodiments in accordance with the present invention, nucleotides is extended or is truncated in the centre of corresponding sequence, can continuously lack 0-3
Individual nucleotides, it is also possible to continuous many 0-3 nucleotides.
According to embodiments of the present invention, the sense primer is SEQ ID NO:1~SEQ ID NO:Nucleotides shown in 13
The sense primer group of sequence composition, the anti-sense primer is SEQ ID NO:14~SEQ ID NO:Nucleotide sequence shown in 17
The anti-sense primer group of composition.
Embodiments in accordance with the present invention, sense primer and/or anti-sense primer are than SEQ ID NO:1~SEQ ID NO:In 17
Corresponding sequence have more 1~3 base, nucleotides more is complementary with the template nucleic acid of position.
It is alleged " by with SEQ ID NO:1~SEQ ID NO:The one-to-one one group of sequence of nucleotide sequence shown in 13
Row " refer to this group of sequence also comprising 13 sequences.Embodiments in accordance with the present invention, every sequence in 13 sequences compares SEQ
ID NO:1~SEQ ID NO:Corresponding sequence in 13 is more or lacks 0~3 nucleotides.For example, there is a sequence in this group of sequence
Than SEQ ID NO:Sequence shown in 1 is more or lacks 0~3 sequence of nucleotides, has another sequence to compare SEQ in this group of sequence
ID NO:Many or few this group of sequence of 0~3 sequence ... of nucleotides of sequence shown in 2 also has a sequence than SEQ ID NO:13
Shown sequence is more or lacks 0~3 sequence of nucleotides.
Embodiments in accordance with the present invention, 5 ' ends of the anti-sense primer and/or 5 ' ends of sense primer include label respectively
Sequence, the sequence label is the sequence bar code being made up of 6~8 nucleotide sequences, wherein, between the sequence bar code at least
There is a nucleotides difference.The primer of the tape label sequence of offer can be to the RNA molecule of each in testing sample or DNA molecular
A sequence label is all added, the sequence label is by tetra- kinds of basic base random combines of ATCG, and the sequence label is mutual
Differ, for example, (being expressed as 8N sequence labels in the embodiment of the present invention) when the base number of sequence label is eight, can obtain
Obtain 109Individual different sequence label combination；When the base number of sequence label is seven, 10 can be obtained8Individual different label sequence
Row combination；When the base number of sequence label is six, 10 can be obtained7Individual different sequence label combination, the label sequence
The base number of row is eight, or seven, or six.
Embodiments in accordance with the present invention, the sense primer also includes SEQ ID NO:20~SEQ ID NO:Shown in 32
Nucleotide sequence, and/or, the anti-sense primer also includes SEQ ID NO:33~SEQ ID NO:Nucleotides sequence shown in 36.
Second aspect, the invention provides a kind of method of acquisition BCR, comprises the following steps：Obtain the core of sample to be tested
Acid；The nucleic acid is expanded using multi-PRC reaction, to obtain multiplexed PCR amplification product, the multi-PRC reaction profit
Carried out with the kit of above-mentioned first aspect present invention or any embodiment.
Embodiments in accordance with the present invention, the testing sample nucleic acid is DNA and/or RNA.
Embodiments in accordance with the present invention, the amount of the nucleic acid is no less than the DNA and/or RNA included in 0.5 cell.
When the nucleic acid for taking testing sample is DNA, the reaction system of multiplex PCR can refer to regular-PCR system to be carried out
Configure or according to the commercially available PCR reaction system kits comprising polymerase；When the nucleic acid for taking testing sample is RNA,
Typically need RNA single strand reverse transcription into cDNA first, with the cDNA chains be again then templated synthesis complementary strand.
Embodiments in accordance with the present invention, the sample to be tested is human peripheral blood single nucleus cell.One of the invention
Embodiment, the RNA in human peripheral blood single nucleus cell is extracted using commercially available RNA extracts kits.
According to embodiments of the present invention, the sample to be tested comes from human leukemia MRD.
Embodiments in accordance with the present invention, when the nucleic acid of sample to be tested is RNA, described use multi-PRC reaction, amplification
The step of testing sample, acquisition multiplexed PCR amplification product, includes：CDNA is obtained with anti-sense primer or sense primer reverse transcription；So
Afterwards with the cDNA as template, corresponding sense primer or anti-sense primer group are added, obtain double-stranded DNA.Further, the reactant
Upstream and downstream primer sets in system are expanded by template of double-stranded DNA, obtain multiplexed PCR amplification product.
Embodiments in accordance with the present invention, in multi-PRC reaction system, 13 sense primer groups of sense primer composition
In, each sense primer equimolar mixing；In 4 anti-sense primer groups of anti-sense primer composition, each anti-sense primer equimolar mixing.
Embodiments in accordance with the present invention, in the system of multi-PRC reaction, template amount is 1~3ug/50ul systems.
Embodiments in accordance with the present invention, the program of the multi-PRC reaction is：
Embodiments in accordance with the present invention, after the multi-PRC reaction terminates, electrophoresis, it is 100- that fragment length is reclaimed in rubber tapping
The DNA fragmentation of 150bp.
Embodiments in accordance with the present invention, gained multiple PCR products are carried out to carry out high-flux sequence after building storehouse, and by life
Thing bioinformatics analysis high-flux sequence result.
The third aspect, the method the invention provides BCR sequencing libraries are obtained, comprises the following steps：Using the present invention the
The method of the acquisition BCR gene orders of two aspects or any embodiment obtains multiplexed PCR amplification product；To the multiplex PCR
Amplified production carries out sequencing library structure, to obtain BCR sequencing libraries.The BCR high-throughput sequencing libraries that the method is obtained can be obtained
The BCR sequences that the length and quantity for obtaining are enriched, are conducive to polymorphism degree analyzing and BCR the clone CDR3 head of district high of BCR sequences
Degree polymorphism distributional analysis.The description of the technical characteristic and advantage of foregoing kit and/or method in any embodiment, together
The method that sample is applicable this aspect of the present invention, will not be repeated here.
Fourth aspect, the method the invention provides being measured to BCR sequences, comprises the following steps：Using the present invention
The method for preparing BCR sequencing libraries of the third aspect or any embodiment obtains BCR sequencing libraries；Text is sequenced to the BCR
Storehouse is sequenced.Sequencing can be carried out in microarray dataset, it is possible to use microarray dataset include but is not limited to the first generation, the second generation
With third generation microarray dataset.The description of the technical characteristic and advantage of foregoing kit and/or method in any embodiment, together
The method that sample is applicable this aspect of the present invention, will not be repeated here.
5th aspect, the invention provides a kind of BCR method for analyzing diversity, including：Using fourth aspect present invention
Or the method being measured to BCR sequences in any embodiment obtains sequencing result；The sequencing result is analyzed,
To obtain the multifarious analysis results of BCR.The technical characteristic and advantage of foregoing kit and/or method in any embodiment
Description, the method for equally applicable this aspect of the present invention will not be repeated here.
Embodiments in accordance with the present invention, the lower machine data based on high-flux sequence platform are entered to people BCR gene C DR3 regions
The comprehensive bioinformatic analysis of row, obtain gene Preferences (usage patterns) of the BCR when VDJ is recombinated, gene
Preference (usage patterns), the CDR3 amino acid sequences of amino acid in combination, junctional diversity information, and CDR3 sequences
The length diversity of row and the characteristic of junction N-terminal base.Exactly these factor quantity of formation are huge and BCR of wide variety
Receptoire.
In a word, based on using the kit comprising above-mentioned multiple PCR primer and the method using the kit, can 1) obtain
Obtained people's BCR sequences；2) the BCR CDR3 sequences of human specific are obtained, the inspection of low copy number B cell clone has been improved particularly
Extracting rate.
Brief description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of genome provided in an embodiment of the present invention and PCR primer；
Fig. 2 is the VDJ recombination analysis results of embodiment of the present invention gained sequence.
Specific embodiment
Material and reagent explanation：
Bone-marrow-derived lymphocyte related leukemia patient：From Shenzhen people's hospital, patient's informed consent.No special explanation,
The reagent that the embodiment of the present invention is used is commercial goods, and the database that the embodiment of the present invention is used is disclosed online data
Storehouse.
SEQ ID NO in the present invention:1~SEQ ID NO:Nucleotides sequence shown in 17 is classified as the primer sequence of design.SEQ
ID NO:18~SEQ ID NO:Nucleotides sequence shown in 19 is classified as the joint sequence used when embodiment Chinese library builds；SEQ
ID NO:20~SEQ ID NO:Nucleotides sequence shown in 36 is classified as the primer sequence used in the embodiment of the present invention.
Wherein, SEQ ID NO：1 is AGAGTCACCATGACCACAG.
SEQ ID NO：2 is AGAGTCACCAKKACCAGGGA.
SEQ ID NO：3 is AGAGTCACCATGACCGAGG.
SEQ ID NO：4 is AGAGTCACCATTACYAGGG.
SEQ ID NO：5 is AGAGTCACGATWACCRCGG.
SEQ ID NO：6 is AGAGTCACCATGACCAGGA.
SEQ ID NO：7 is ACCAGGCTCACCATYWCCAAG.
SEQ ID NO：8 is GGCCGATTCACCATCTCMA.
SEQ ID NO：9 is CGAGTCACCATRTCMGTAG.
SEQ ID NO：10 is CAGCCGACAAGTCCATCA.
SEQ ID NO：11 is AGTCGAATAACCATCAACCC.
SEQ ID NO：12 is GACGGTTTGTCTTCTCCTTG.
SEQ ID NO：13 is AGAGTCACCATGACCACAG.
SEQ ID NO：14 is CTGAGGAGACRGTGACCAGGGTG.
SEQ ID NO：15 is CTGAAGAGACGGTGACCATTGTC.
SEQ ID NO：16 is CTGAGGAGACGGTGACCAGGGT.
SEQ ID NO：17 is TGAGGAGACGGTGACCGTGGTC.
SEQ ID NO：18 areCAGACGTGTGCTCTTCCGATCTAG。
SEQ ID NO：19 areCTACACGACGCTCTTCCGATCT。
Specifically, primer of the present invention is following (underscore part is sequencing company joint sequence)：
The multiple PCR primer sequence of table 1.
Note, it will be appreciated by persons skilled in the art that nucleotides abbreviation is as follows：R=A/G, Y=C/T, M=A/C, K=
G/T, S=C/G, W=A/T, H=A/C/T, B=C/G/T, V=A/C/G, D=A/G/T, N=A/C/G/T.
Design primer：Carry out comparing analysis for all of V and J genes of BCR, using the Hes of Oligo 7.0
MFEprimer-2.0 is analyzed to primer dimer and stem ring mispairing, is set in the CDR3 areas upstream (i.e. FR3 areas) of BCR
Sense primer, reverse primer is designed for J downstream of gene, expands CDR3 regional sequences.
The primer sets that the present embodiment is provided cover most of VDJ recombinant fragments.Because the sequence variation of very little will cause
Primer expanding effect is significantly reduced, and inventor devises 3 groups of multiplex PCRs for the different sections in different purpose BCR regions respectively
Primer sets, by after 3 groups of preliminary experiment screenings, the present invention have chosen the optimal primer sets of expanding effect, as shown above.
Embodiment 1
The embodiment of the present invention 1 provides a kind of preparation method of b lymphocyte receptor (BCR) DNA sample, including following step
Suddenly：
(1) collect fresh peripheral blood sample it is each 10 milliliters (ml), by LymphoPrep kits (Axis-shield,
Cat.No.AS1114544 UK) specification operation, obtain relatively pure PBMC；
(2) using PureLink Genomic DNA Mini Kit (Life Technology, Cat.No:K1820-00)
Kit extraction step (1) gained cell genomic DNA, and with Nanodrop2000 (Thermo) measure DNA concentration and
Purity, then preserves genomic DNA.
DNA extracts electrophoresis result, and (genomic DNA fragment is referring to swimming lane 1-2 as shown in Fig. 1-a；M is DNA Marker).
Embodiment 2
The multiple PCR primer that the embodiment of the present invention 2 provides a kind of use Minimal Residual Disease of Leukemia stove BCR libraries builds
The method of Minimal Residual Disease of Leukemia stove BCR high-throughput sequencing libraries, comprises the following steps：
It is amplification template with the gained genomic DNA of embodiment 1, takes BCR primers, then using QIAGEN companies Multiplex
PCR kit (article No.：206143) multiplex PCR system, is configured by kit specification, wherein, BCR primers include sense primer
And anti-sense primer, the sense primer is SEQ ID NO:1~SEQ ID NO:The upstream of the nucleotide sequence composition shown in 13
Primer sets, the anti-sense primer is SEQ ID NO:14~SEQ ID NO:The anti-sense primer of the nucleotide sequence composition shown in 17
Group.
Each sense primer equimolar mixing, total primer concentration is 10 micromoles, and each anti-sense primer equimolar mixing, primer is total
Concentration is 10 micromoles, and template amount can be adjusted, and 3ug is used in the present embodiment.
Survey is sent for convenience, unless otherwise noted, when the embodiment of the present invention carries out multiplex PCR, respectively in sense primer with
Trip primer adds sequence measuring joints, specially：The sense primer of illumina sequencing companies is connected respectively at 5 ' ends of sense primer
Joint sequence (such as SEQ ID NO:Nucleotide sequence shown in 18), illumina sequencings are connected respectively at 5 ' ends of anti-sense primer
Anti-sense primer joint sequence (such as SEQ ID NO of company:Nucleotide sequence shown in 19), specific steps are high with reference to illumina
Flux sequencing library builds specification；
Again by the condition setting PCR instrument device program of following multiplex PCRs, multiplex PCR is carried out：
After PCR terminates, 4 DEG C preserve PCR primer and electrophoresis detection, as a result as shown in Fig. 1-b, are cut about under ultraviolet
(purpose fragment is referring to swimming lane 1-3 for the purpose fragment of 250bp or so；M is DNA Marker).
The CDR3 fragments of fragment length about 250bp are selected, rubber tapping is reclaimed, and obtains CDR3 fragments after purification, glue reclaim
Step uses QIAGEN companies QIAquick gel purification kits, and routinely laboratory operation is carried out；Nanodrop 2000 is tested
DNA concentration, and send the company to carry out high-flux sequence (being sequenced using Illumina hiseq2000,2*100pair-end).
After primer sets of the invention and multiplex PCR structure storehouse, high-flux sequence obtains about million sequences.To survey
Sequence result carries out bioinformatics statistical analysis, and (bioinformatic analysis are using southern University of Science and Technology's online software Immune
Repertoire Analysis Pipeline (iRAP, http：//www.sustc-genome.org.cn/irap/), part
Comparison result is as shown in table 2 below.Wherein, table 2 is that CDR3 unique clone quantity and distribution, including table 2-1 and table 2-2.
In table 2-1, Total reads number：Sample always compares reads numbers；Immune sequences numebr：
Compare the reads numbers to target area；Unknown sequences numebr：Fail the reads successfully compared in database
Number；productive sequences number：It is identified as TCR β chains and the reads numbers that can effectively translate；Non_
productive sequences number：It is identified as TCR β chains but the reads numbers that can not effectively translate；In-frame
sequences number：It is identified as TCR β chains and also in the reads numbers of normal reading inframe；Out-of_frame
sequences number：It is identified as TCR β chains but there occurs the reads numbers of frameshift mutation；Total CDR3 sequences
number：All sequence sums that can detect CDR3；Unique CDR3 nt sequences number：All of CDR3
Sequence de-redundancy after base sequence species number；Unique CDR3 aa sequences number：The sequence of all of CDR3
Amino acid sequence species number after row de-redundancy.
Table 2-1.CDR3 unique clone quantity and distribution
| Total reads number | 2031308 | 1987751 | 1438302 | 783456 | 960837 | 1073258 |
| immune sequences number | 1997429 | 1895548 | 1373941 | 757634 | 921853 | 1027270 |
| Unknown seouences numebr | 33879 | 92203 | 64361 | 25822 | 38984 | 45988 |
| productive sequences number | 1665974 | 1485444 | 1069650 | 617752 | 683431 | 630666 |
| Non_productive sequences number | 331455 | 410104 | 304291 | 139882 | 238422 | 396604 |
| In-frame sequences number | 1742745 | 1554969 | 1130464 | 648423 | 751370 | 823125 |
| Out-of_frame sequences number | 249753 | 336743 | 240804 | 107677 | 168555 | 201943 |
| Total CDR3 sequences number | 1247543 | 1458146 | 1048736 | 607143 | 668171 | 615392 |
| Unique cdr5 nt sequences number | 97346 | 68127 | 103347 | 56940 | 76489 | 60181 |
| Unique cdr3 aa sequences number | 85832 | 56874 | 90119 | 50791 | 67745 | 52124 |
Table 2-2.CDR3 unique clone quantity and distribution
| CDR3 ID | CDR3 Sequence(nt) | CDR3 Seauence (aa) | Reads unique | Ratio |
| >CG2_uniquecdr3nt_1 | AGCGTGAGAGCGGTACAAGA GACCCAGTAC | SVRAVQETQY | 11553 | 6.54% |
| >CG2_uniquecdr3nt_2 | GCCACCAGTGATTTGCAGGG GGATGCCGGGGAGCTGTTT | ATSDLQGDAGELF | 6669 | 3.78% |
| >CG2_uniquecdr3nt-3 | GCCAGCACTGGTCACCTAAA TGAAAAACTGTTT | ASTGHLNEKLF | 6439 | 3.64% |
| >CG2_uniquecdr3nt_4 | GCCAGCAGCTTAGAGGCGCA TGCGAACACCGGGGAGCTGT TT | ASSLEAHANTGELF | 5730 | 3.24% |
| >CG2_uniquecdr3nt_5 | GCTAGTGGTGCAGGGTTACT CTGGACTGAAGCTTTC | ASGAGLLWTEAF | 5673 | 3.21% |
| >CG2_uniquecdr3nt_6 | GCCACCAGCAGAGATACGTT GGCGGGGGGCCGGGGGGATA CGCAGTAT | ATSRDTLAGGRGDTQY | 1907 | 1.08% |
| >CG2_uniquecdr3nt_7 | GCCAGCAGCTACACCTCGAA CACCGGGGAGCTGTTT | ASSYTSNTGELF | 1743 | 0.99% |
| >CG2_uniquecdr3nt_8 | GCCAGCAGCTTATCCCAGGG CCAGGACACTGAAGCTTTC | ASSLSQGQDTEAF | 1154 | 0.65% |
| >CG2_uniquecdr3nt_9 | GCCACCAGTTGGGGCACCAC CACCTACAATGAGCAGTTC | ATSWGTTTYNEQF | 1035 | 0.59% |
| >CG2_uniquecdr3nt_10 | GCCAGCAGCGAGCTGACAGG GGGGGGGCTCGAGCAGTAC | ASSELTGGGLEQY | 934 | 0.53% |
By comparison and bioinformatic analysis, every sequence information of CDR3 sequences, amino acid can be exactly known
Information, bar number and proportion.Compared by BCR and analyzed, present invention obtains high-flux sequence sequence C DR3 representativenesses gram
Grand statistic analysis result, as a result as shown in Fig. 2 Fig. 2 is applied in combination situation for BCR CD3 area V-J.As shown in Figure 2, by this
The primer of invention is obtained in nearly white ten thousand BCR sequences, and the CDR3 sequences of unique are more than 104Bar.
The above results show that building Minimal Residual Disease of Leukemia stove BCR libraries using the method for the present invention can cover IGH
The diversity information of gene.
Presently preferred embodiments of the present invention is the foregoing is only, is not intended to limit the invention, it is all in essence of the invention
Any modification, equivalent and improvement made within god and principle etc., should be included within the scope of the present invention.To the greatest extent
Pipe above embodiments of the present invention have been shown and described, it is to be understood that above-mentioned implementation method be it is exemplary, no
It is understood that to be limitation of the present invention, one of ordinary skill in the art within the scope of the invention can be to above-mentioned implementation method
It is changed, changes, replacing and modification.
Sequence table（SEQUENCE LISTING）
<110>Big desert gene biological Science and Technology Ltd. of Shenzhen
<120>BCR diversity detection kit and application
<130> PI2015010_1
<160> 36
<170> PatentIn version 3.5
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<223>K is g or t/u
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<211> 19
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<221> misc_feature
<222> (1)..(19)
<223>Primer
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agagtcacca tgaccgagg 19
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<212> DNA
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<221> misc_feature
<222> (1)..(19)
<223>Primer
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<223>Y is t/u or c
<400> 4
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<213>Artificial sequence
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<221> misc_feature
<222> (1)..(19)
<223>Primer
<220>
<221> misc_feature
<222> (12)..(12)
<223>W is a or t/u
<220>
<221> misc_feature
<222> (16)..(16)
<223>R is g or a
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agagtcacga twaccrcgg 19
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<211> 19
<212> DNA
<213>Artificial sequence
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<221> misc_feature
<222> (1)..(19)
<223>Primer
<400> 6
agagtcacca tgaccagga 19
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(21)
<223>Primer
<220>
<221> misc_feature
<222> (15)..(15)
<223>Y is t/u or c
<220>
<221> misc_feature
<222> (16)..(16)
<223>W is a or t/u
<400> 7
accaggctca ccatywccaa g 21
<210> 8
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(19)
<223>Primer
<220>
<221> misc_feature
<222> (18)..(18)
<223>M is a or c
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ggccgattca ccatctcma 19
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<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(19)
<223>Primer
<220>
<221> misc_feature
<222> (12)..(12)
<223>R is g or a
<220>
<221> misc_feature
<222> (15)..(15)
<223>M is a or c
<400> 9
cgagtcacca trtcmgtag 19
<210> 10
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(18)
<223>Primer
<400> 10
cagccgacaa gtccatca 18
<210> 11
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(20)
<223>Primer
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agtcgaataa ccatcaaccc 20
<210> 12
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(20)
<223>Primer
<400> 12
gacggtttgt cttctccttg 20
<210> 13
<211> 19
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(19)
<223>Primer
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agagtcacca tgaccacag 19
<210> 14
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(23)
<223>Primer
<220>
<221> misc_feature
<222> (11)..(11)
<223>R is g or a
<400> 14
ctgaggagac rgtgaccagg gtg 23
<210> 15
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(23)
<223>Primer
<400> 15
ctgaagagac ggtgaccatt gtc 23
<210> 16
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(22)
<223>Primer
<400> 16
ctgaggagac ggtgaccagg gt 22
<210> 17
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(22)
<223>Primer
<400> 17
tgaggagacg gtgaccgtgg tc 22
<210> 18
<211> 24
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(24)
<223>Joint
<400> 18
cagacgtgtg ctcttccgat ctag 24
<210> 19
<211> 22
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(22)
<223>Joint
<400> 19
ctacacgacg ctcttccgat ct 22
<210> 20
<211> 46
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(46)
<223>Primer
<400> 20
cagacgtgtg ctcttccgat ctagctacac gacgctcttc cgatct 46
<210> 21
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(44)
<223>Primer
<220>
<221> misc_feature
<222> (35)..(35)
<223>K is g or t/u
<220>
<221> misc_feature
<222> (36)..(36)
<223>K is g or t/u
<400> 21
cagacgtgtg ctcttccgat ctagagagtc accakkacca ggga 44
<210> 22
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(43)
<223>Primer
<400> 22
cagacgtgtg ctcttccgat ctagagagtc accatgaccg agg 43
<210> 23
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(43)
<223>Primer
<220>
<221> misc_feature
<222> (39)..(39)
<223>Y is t/u or c
<400> 23
cagacgtgtg ctcttccgat ctagagagtc accattacya ggg 43
<210> 24
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(43)
<223>Primer
<220>
<221> misc_feature
<222> (36)..(36)
<223>W is a or t/u
<220>
<221> misc_feature
<222> (40)..(10)
<223>R is g or a
<400> 24
cagacgtgtg ctcttccgat ctagagagtc acgatwaccr cgg 43
<210> 25
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(43)
<223>Primer
<400> 25
cagacgtgtg ctcttccgat ctagagagtc accatgacca gga 43
<210> 26
<211> 45
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(45)
<223>Primer
<220>
<221> misc_feature
<222> (39)..(39)
<223>Y is /u or c
<220>
<221> misc_feature
<222> (40)..(10)
<223>W is a or t/u
<400> 26
cagacgtgtg ctcttccgat ctagaccagg ctcaccatyw ccaag 45
<210> 27
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(43)
<223>Primer
<220>
<221> misc_feature
<222> (42)..(42)
<223>M is a or c
<400> 27
cagacgtgtg ctcttccgat ctagggccga ttcaccatct cma 43
<210> 28
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(43)
<223>Primer
<220>
<221> misc_feature
<222> (36)..(36)
<223>R is g or a
<220>
<221> misc_feature
<222> (39)..(39)
<223>M is a or c
<400> 28
cagacgtgtg ctcttccgat ctagcgagtc accatrtcmg tag 43
<210> 29
<211> 42
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(42)
<223>Primer
<400> 29
cagacgtgtg ctcttccgat ctagcagccg acaagtccat ca 42
<210> 30
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(44)
<223>Primer
<400> 30
cagacgtgtg ctcttccgat ctagagtcga ataaccatca accc 44
<210> 31
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(44)
<223>Primer
<400> 31
cagacgtgtg ctcttccgat ctaggacggt ttgtcttctc cttg 44
<210> 32
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(43)
<223>Primer
<400> 32
cagacgtgtg ctcttccgat ctagagagtc accatgacca cag 43
<210> 33
<211> 45
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(45)
<223>Primer
<400> 33
ctacacgacg ctcttccgat ctctgaggag acrgtgacca gggtg 45
<210> 34
<211> 45
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(45)
<223>Primer
<400> 34
ctacacgacg ctcttccgat ctctgaagag acggtgacca ttgtc 45
<210> 35
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(44)
<223>Primer
<400> 35
ctacacgacg ctcttccgat ctctgaggag acggtgacca gggt 44
<210> 36
<211> 44
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<222> (1)..(44)
<223>Primer
<400> 36
ctacacgacg ctcttccgat cttgaggaga cggtgaccgt ggtc 44
Claims (10)
1. a kind of to detect the multifarious kits of BCR, the kit includes one group of multiple PCR primer, it is characterised in that described
Multiple PCR primer includes sense primer and anti-sense primer,
The sense primer by with SEQ ID NO:1~SEQ ID NO:The one-to-one one group of sequence of nucleotide sequence shown in 13
Row composition, the sequence in the sense primer is than SEQ ID NO:1~SEQ ID NO:Corresponding sequence in 13 is more or few 0~3
Individual nucleotides,
The anti-sense primer by with SEQ ID NO:14~SEQ ID NO:One-to-one one group of nucleotide sequence shown in 17
Sequence is constituted, and the sequence in the anti-sense primer is than SEQ ID NO:14~SEQ ID NO:Corresponding sequence in 17 is more or few 0
~3 nucleotides.
2. the kit of claim 1, it is characterised in that the sense primer is SEQ ID NO:1~SEQ ID NO:13 institutes
The sense primer group of the nucleotide sequence composition for showing, the anti-sense primer is SEQ ID NO:14~SEQ ID NO:Shown in 17
The anti-sense primer group of nucleotide sequence composition.
3. the kit of claim 1 or 2, it is characterised in that the sense primer also includes SEQ ID NO:20~SEQ ID
NO:Nucleotide sequence shown in 32, and/or,
The anti-sense primer also includes SEQ ID NO:33~SEQ ID NO:Nucleotide sequence shown in 36.
4. a kind of method of acquisition BCR gene orders, it is characterised in that comprise the following steps：
Obtain the nucleic acid of sample to be tested；
The nucleic acid is expanded using multi-PRC reaction, to obtain multiplexed PCR amplification product, the multi-PRC reaction profit
Carried out with the kit described in claim any one of 1-3.
5. the method for claim 4, it is characterised in that the testing sample nucleic acid is DNA and/or RNA；
Optionally, the amount of the nucleic acid is no less than DNA and/or RNA contained in 0.5 cell of people；
Optionally, the sample to be tested is human peripheral blood single nucleus cell；
Optionally, the sample to be tested comes from human leukemia MRD.
6. the method for claim 4 or 5, it is characterised in that when testing sample nucleic acid is RNA, the use multi-PRC reaction
The nucleic acid is expanded, the step of to obtain multiplexed PCR amplification product, including：
Reverse transcription is carried out by reverse transcription primer of the sense primer or the anti-sense primer, cDNA products are obtained；
With the cDNA products as template, add corresponding anti-sense primer or sense primer to carry out the multi-PRC reaction, obtain
Obtain the multiplexed PCR amplification product.
7. a kind of method for preparing BCR sequencing libraries, it is characterised in that comprise the following steps：
Multiplexed PCR amplification product is obtained using claim 4-6 either method；
Sequencing library structure is carried out to the multiplexed PCR amplification product, to obtain BCR sequencing libraries.
8. a kind of method of measure BCR gene orders, it is characterised in that including：
BCR sequencing libraries are obtained using the method for claim 7；
The BCR sequencing libraries are sequenced.
9. it is a kind of to analyze the multifarious methods of BCR, it is characterised in that including：
Using the method for claim 8, sequencing result is obtained；
The sequencing result is analyzed, to obtain the multifarious analysis results of BCR.
10. any kits of claim 1-3 obtain BCR gene orders and/or detection BCR diversity in purposes.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710085553.5A CN106834472A (en) | 2015-08-14 | 2015-08-14 | BCR diversity detection kit and application |
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| CN201510500391.8A CN105063032A (en) | 2015-08-14 | 2015-08-14 | Multiple PCR primers and method for constructing leukemia minimal residual disease BCR library based on high-flux sequencing |
| CN201710085553.5A CN106834472A (en) | 2015-08-14 | 2015-08-14 | BCR diversity detection kit and application |
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Cited By (2)
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| CN107267629A (en) * | 2017-07-13 | 2017-10-20 | 武汉赛云博生物科技有限公司 | A kind of method of the B cell minimal residual detection of leukaemia |
| CN112301099A (en) * | 2020-11-30 | 2021-02-02 | 南方科技大学 | Primer group for amplifying B lymphocyte immune repertoire and application thereof |
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| CN106834472A (en) * | 2015-08-14 | 2017-06-13 | 深圳市瀚海基因生物科技有限公司 | BCR diversity detection kit and application |
| RU2638800C2 (en) * | 2015-12-29 | 2017-12-15 | Федеральное государственное бюджетное учреждение науки институт биоорганической химии им. академиков М.М. Шемякина и Ю.А. Овчинникова Российской академии наук (ИБХ РАН) | Method for tumour b lymphoblasts availability markers determination |
| CN107345241A (en) * | 2016-05-12 | 2017-11-14 | 眭维国 | B cell antigen receptor H chains CDR3 processing method |
| CN106119251A (en) * | 2016-06-03 | 2016-11-16 | 刘鹏飞 | BCR heavy chain CDR3 leukemia is caused a disease sequence and screening technique and application |
| CN107955831A (en) * | 2016-10-13 | 2018-04-24 | 深圳华大基因研究院 | The label and lymphocyte quantitative detecting method quantitatively detected for lymphocyte |
| CN106957906B (en) * | 2016-12-23 | 2020-04-28 | 孙涛 | Primer combination and kit applied to high-throughput sequencing detection of T cell leukemia minimal residual disease |
| CN108823306A (en) * | 2017-05-03 | 2018-11-16 | 杭州赫玛生物科技有限公司 | The system and method for detecting leukaemia cell |
| EP3824104A1 (en) * | 2018-07-18 | 2021-05-26 | Life Technologies Corporation | Compositions and methods for immune repertoire sequencing |
| CN109680062B (en) * | 2018-12-18 | 2022-12-02 | 杭州艾沐蒽生物科技有限公司 | Method for detecting minimal residual disease MRD |
| CN111261226B (en) * | 2020-03-12 | 2020-10-27 | 江苏先声医学诊断有限公司 | NGS-based automatic sequencing analysis method and device for minimal residual lesions |
| CN114807123B (en) * | 2021-01-29 | 2023-12-01 | 深圳华大基因科技服务有限公司 | Design and connection method of amplification primers of DNA molecules |
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Also Published As
| Publication number | Publication date |
|---|---|
| WO2017028752A1 (en) | 2017-02-23 |
| CN105063032A (en) | 2015-11-18 |
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